Regulated reactions are often those which are essentially irreversible (for energetic reasons)

Regulation often takes place (Fig. 2.2):

Early in a linear pathway

At branch points

Reciprocally, at bi-directional points, with a different enzyme for the forward and reverse directions.

Cycles between organs In addition to cellular compartmentalization being an important feature of metabolic pathways, there is also a sharing of metabolic load between organs of the body. One good example of this is the Cori cycle (see Fig. 2.3).

These types of inter-organ cycles have multiple levels of potential control, including:

Small changes in plasma hormone levels often have large effects on cell functions due to amplification cascades.

Fig. 2.2 Metabolic control: regulation.

Fig. 2.3 The Cori cycle.

P.97 P.98 Oxidation and reduction Generally speaking:

Breakdown (catabolic) reactions involve oxidation

Synthetic (anabolic) reactions involve reduction

Rather than being directly linked, intermediate molecules store/ donate the electrons. For example, in glycolysis, glyceraldehydes-3-phosphate is oxidized while the intermediate NAD+ is reduced to NADH.

There are three main biological intermediates (Figs. 2.4, 2.5):

Nicotinamide adenine dinucleotide (NAD+)

Flavin adenine dinucleotide (FAD)

Nicotinamide adenine dinucleotide phosphate (NADP+).

Compartmentalization allows controlled oxidation and reduction reactions to occur in the same cell.

Most of the NAD+ and FAD are unreduced and in the mitochondria (ideal for oxidative reactions)

In contrast, most NADP+ is in the reduced form, NADPH in the cytosol, where it participates in reactions involving reduction.

Fig. 2.5 Structure of the oxidized form of flavin adenine dinucleotide (FAD).

P.99 P.100 Body energy supplies Food intake (OHCM6 p.208) is not a continuous process, and so the body must be able to store energy. Energy can be stored in a variety of ways, listed below in order of use:

Carbohydrate

Glucose in plasma (3 litres with an average concentration of 5mM)

Glucose is stored as the polymer, glycogen, in all cells but the two major sites are:

Liver (10% of total tissue mass): used to maintain blood glucose during short periods of fasting; enough stores for about 24 hours at rest

Skeletal muscle (2% of muscle mass): only used by muscle itself during exercise.

Lipid (fat)

The majority of lipid is stored in adipose tissue (fat cells)

Fat accounts for about 15kg of a typical 70kg man

Fat is a highly compact energy store (over 300-fold more energy in body fat stores than in liver glycogen)

Enough fat stores for about 3 months

Protein

Protein is not a classical energy store but can be used in extreme starvation conditions when other stores have been exhausted

Skeletal muscle is the major store of mobilizable protein

Loss of protein from heart, kidney, and liver compromises their function and will lead to death.

About 35g minimum required per day to maintain nitrogen balance (i.e. to ingest as much nitrogen as we excrete)

Carbohydrate-free diets (such as the âAtkins dietâ) work by tricking the body into starvation mode (pp.166â7), with energy obtained from protein and fat metabolism due to the prevailing glucagon signal. As with all diets, for it to be effective, energy intake must be less than energy expenditure.

P.101 P.102 Central Metabolic Pathways Tricarboxylic acid (TCA) cycle and its control The TCA cycle is the common pathway for the oxidation of fuel molecules.

Also known as the Krebs cycle (after its discoverer) or the citric acid cycle

It is a cyclic pathway: intermediates are regenerated so that net amounts of each remain the same after each turn of the cycle

Intermediates present in relatively small amounts, and essentially play a catalytic role

Many intermediates are starting points for biosynthetic pathways. Anapleurotic reactions fill up the cycle to replace any of the intermediates used in this way.

The TCA cycle

The reactions of the TCA cycle take place in the mitochondrial matrix

Pyruvate enter the mitochondria on a specific transporter in the IMM

Entry point into the TCA cycle is the compound, acetyl-CoA

Acetyl-CoA is formed from pyruvate (the end-point of glycolysis; pp.128â30) by the link reaction in Fig. 2.6.

Acetyl-CoA can also come from fatty acid breakdown or the carbon skeletons of amino acids

PDH is inhibited directly by high levels of acetyl-CoA and NADH, and indirectly by ATP, acetyl-CoA, and NADH (all of which activate PDH kinase, which phosphorylates PDH and inactivates it; PDH kinase is activated by PDH substrates pyruvate, CoA-SH and NAD+).

Regulation of TCA cycle

The rate of TCA cycling matches the cellular demand for ATP, and not the availability of substrates

The main regulated enzymes are isocitrate dehydrogenase (inhibited by ATP, NADH; activated by ADP) and Î±-ketoglutarate dehydrogenase (inhibited by ATP, NADH, succinyl CoA)

TCA cycle is therefore inhibited when the cell has no need for further ATP synthesis, and activated when it needs to make more ATP

The regulated TCA enzymes are also activated by a rise in intra-mitochondrial Ca2+

The rise in Ca2+ could be caused by adrenaline (âflight or fightâ response) or increased muscle contraction. Both of these situations will increase ATP consumption, so the TCA cycle is stimulated to increase ATP synthesis.

The large protein complexes will only move relatively slowly in the lipid bilayer

Coenzyme Q and cytochrome C are small, highly mobile electron carriers that transport electrons from one complex to another

The reaction centres have increasing redox potential

Three of the four complexes are also proton pumps: for each pair of electrons, complex I extrudes 4H+ from the matrix; complex III, 4H+; and complex IV, 2H+. Complex II does not pump protons when it transfers electrons from FADH2 to coenzyme Q

Thus, for each NADH oxidized, 10H+ are extruded; and, for each FADH2, 6H+

The ultimate electron acceptor is molecular oxygen, which is reduced to water.

Although most NADH is formed in the mitochondria during the TCA cycle (p.102), it is also formed cytoplasmically e.g. in glycolysis (pp.128â30).

There is no direct pathway for NADH to cross the IMM to enter the ETC. If there was, this would destroy the distinct oxidative/reductive compartments of the cell

NADH can effectively cross the membrane by means of the malate/aspartate shuttle (Fig. 2.9)

When cytoplasmic (NADH) is low, the glycerol-3-phosphate shuttle may be used (Fig. 2.10)

Electrons enter the ETC at the level of FADH2 and so get less ATP per original NADH than with the malate/aspartate shuttle.

Fig. 2.8 The electron transport chain.

Fig. 2.9 Malateâaspartate shuttle.

Fig. 2.10 Glycerol-3-phosphate shuttle.

P.105 P.106 ATP synthesisâthe chemiosmotic theory The chemiosmotic theory (Fig. 2.11) was proposed by Peter Mitchell1 in 1961 and is based on the following premise:

The inner mitochondrial membrane (IMM) is impermeable to protons, hence the mitochondrial matrix is a closed environment

The proton pumping of the ETC complexes (p.104) leads to the generation of the proton motive force (PMF; total magnitude of 0.224V)

The PMF provides the energy for ATP synthesis

Evidence: agents that collapse this PMF inhibit ATP formation

These compounds are weak lipophilic acids that carry protons across the IMM e.g. 2,4-dinitrophenol and salicylic acid

Respiratory control Electrons cannot flow through the ETC unless ADP is simultaneously phosphorylated to ATP.

The most significant controlling factor for electron flow is the availability of ADP for conversion to ATP. In this way, the ADP concentration exercises what is known as ârespiratory controlâ.

Hypothesis for the evolution of mitochondria It has been proposed that mitochondria were originally free-living bacteria which became incorporated into cells in a symbiotic relationship. This idea is supported by the fact that bacteria also use a PMF to drive uptake of nutrients across their cell wall.

Some antibiotics are proton ionophores that kill bacteria by collapsing their PMF. One such example is the topical antifungal, Nystatin.

Fig. 2.11 Generation of ATP in mitochondria by the chemiosmotic mechanism.

P.107 P.108 ATP synthesisâuses of the proton motive force The proton motive force (PMF) across the inner mitochondrial membrane (IMM) can be used to drive a number of processes. 1. ATP synthesis The impermeability of the IMM to protons, except through the protein responsible for ATP synthesis, is a key feature of the chemiosmotic theory.

This protein is known as the F0F1-ATPase, ATP synthase, or complex V (Fig. 2.12)

The F0 subunit is an integral membrane protein which forms a proton channel

F1 is a complex (Î±3, Î²3, Î³, Î´, and Îµ) that has the catalytic site for ATP synthesis

The F0 and F1 subunits are functionally linked, such that protons can only flow when ATP is being synthesized (dependent on [ADP] = ârespiratory controlâ).

Mechanism of ATP synthesis The movement of protons through the F0 subunit induces the F1 subunit to physically rotate.

This is proposed to propel the binding sites through their different transition states of loose (ADP+Pi), tight (ADP+Pi), and ATP release. Therefore takes three protons to make one ATP (Fig. 2.13).

The F1 subunit can be dissociated from the F0 subunit by protease activity.

When not linked to the F1, it can act as an ATPase

ATP hydrolysis will drive the rotation of the F1 subunit. This is shown experimentally by attaching a fluorescent actin filament and seeing it rotate (âthe worldâs smallest motorâ).

2. Inner membrane transport The proton gradient is also used to drive the movement of compounds through specific transporters in the IMM.

Most ATP is made in the mitochondrial matrix, yet is needed in the cytoplasm; conversely, most ADP is formed in the cytoplasm, but regenerated in the matrix

An obligatory ATP/ATP exchanger (the adenosine nucleotide translocase, ANT) is present in the IMM. Although not proton-coupled, due to the fact that ATP is more negative than ADP (4- vs. 3-), it is driven by the membrane potential component of the PMF

Pi is also required in the matrix for ATP synthesis (although most will be released from ATP hydrolysis in the cytoplasm)

There is a H+/Pi co-transporter in the IMM which effectively means that each ATP formed uses 4 protons. Hence 1 NADH = 2.5 ATP, 1 FADH2 = 1.5 ATP

Pyruvate needs to cross the IMM to enter the TCA cycle

There is a IMM pyruvate/H+ co-transporter

Mitochondria also take up Ca2+ in response to a rise in intracellular levels

Uptake will be electrogenically favourable due to the PMF

Plays a part in regulating the TCA cycle (p.102).

P.109 3. Thermogenesis in brown adipose tissue So far, it has been stressed that the only natural route by which the PMF can be dissipated is through the F0F1-ATPase and the synthesis of ATP.

The only tissue for which this is not true is brown adipose tissue (âbrown fatââbrown due to its high mitochondria content)

There is an uncoupling protein that allows the PMF to be dissipated without making ATP

The energy is released as heat

This is important in neonates who cannot shiver to generate heat.

Fig. 2.12 F2F0 ATP synthase.

Fig. 2.13 The catalytic sites of ATP synthase as proposed in the Boyer model: (a) the changes that occur in a single site of one Î² subunit of F2 during the synthesis of ATP; (b) the three Î² subunits work in a co-operative manner and the conversion in one site are co-ordinated with the other two sites.

P.110 ATP synthesis control The daily turnover of ATP in the average 70kg man is approximately 40kg, yet cells contain relatively little ATP at any one timeâit cannot be stored and ATP molecules have a half-life in the order of seconds. Therefore, ATP production must match usage. Intracellular (ATP) remains virtually constant. Relative concentrations of:

ATPâhigh

ADPâlow

AMPâvery low.

ADP and AMP as controls of ATP synthesis

ADP controls the rate of ATP synthesis at a mitochondrial level through the process of respiratory control (p.106). Unless there is ADP to make into ATP the ETC does not run

AMP is an important intracellular signal. As the normal intracellular concentration is very low, cells are very sensitive to even a small change

The acyl carnitine is reconverted to acyl-CoA and free carnitine in the mitochondrial matrix

Reaction catalysed by carnitine acyl transferase II

A number of diseases are linked to carnitine, acyl carnitine translocase, or acyl carnitine transferase deficiencies

Carnitine deficiency leads to muscle weakness during long-term exercise (when fatty acids are an important source of energy)

Heart and kidney are also affected as they use fatty acids for the majority of their energy supply

Symptoms range from mild muscle cramps to severe weakness and even death.

Once in the mitochondrial matrix, acyl-CoA can be oxidized by the process known as Î²-oxidation (Fig. 2.15).

Four-step cyclic reaction removes a C2 subunit in the form of acetyl-CoA. This can enter the TCA cycle ââ ATP

There are different isoenzymes for reaction 1 depending on the length of the fatty acid being metabolized: very long-chain acyl-CoA dehydrogenase (VLCAD), long-chain (LCAD), medium-chain (MCAD), and short-chain (SCAD).

Fig. 2.14 Mechanism of transport of long chain fatty acyl groups into mitochondria where they are oxidized in the mitochondrial matrix.

Fig. 2.15 One round of the four reactions of B-oxidation by which a fatty acyl-CoA is shortened by two carbon atoms with the production of a molecule of acetyl-CoA.

P.115 P.116Not all fatty acids in our diet are of an even chain length.

Although animals have even chain lengths (i.e. C2n), plants have an odd number of fatty acids

Î²-oxidation eventually leaves a C3 unit (propionyl CoA). This is converted into the TCA cycle intermediate, succinyl-CoA.

Fatty acids can have differing degrees of saturation.

One extra enzyme is required for monounsaturated fatty acid

Normal rounds of Î²-oxidation occur until there is a cis-double bond between the C3 and C4 atoms

An isomerase then rearranges the C = C bond so that it is trans-double bond between C2 and C3

This has formed the trans-enoyl-CoA compound on the Î²-oxidation pathway, which can continue as normal

Any polyunsaturated fatty acid requires two extra enzymes, the isomerase plus a reductase

Î²-oxidation rounds occur with the help of the isomerase until a fatty acid chain with a -C=C-C=C- (trans-double bond between C4 and C5 and cis-double bond between C2 and C3) is formed after the fatty acyl-CoA dehydrogenase step of Î²-oxidation

This cannot be processed further without a reductase enzyme

The reductase utilizes NADPH to reduce this to -C-C=C-C- (trans-double bond between C3 and C4

This can then be isomerized to the trans-enoyl CoA (i.e. cis-double bond between C2 and C3) and metabolized (as above for a monosaturated fatty acid).

Diseases of fatty acid oxidation

Known to be inherited diseases related to deficiencies in all of the acyl-CoA dehydrogenases

Best characterized is deficiency in medium-chain acyl-CoA dehydrogenase (MCAD)

Thought to be one of the most common inborn errors of metabolism

Symptoms include lethargy, vomiting, and often coma after fasting for more than 12 hours

Ketogenesis is blocked in liver by lack of Î²-oxidation of fatty acids

This in turn slows gluconeogenesis

Failure to be able to metabolize fat in muscle causes increase use of glucose, exasperating the hypoglycaemia

Medium-chain fatty acids metabolized by alternative pathways and excreted in urine (the disease can be diagnosed by urine analysis

Disorder can be managed by avoiding fasting

May be the cause of some cases of sudden infant death syndrome.

P.117 P.118 Biosynthesis by the liver During times of plenty, the body will store energy. After the glycogen stores have been replenished (to 10% of liver weight), the liver switches to fat biosynthesis. Both excess sugars and amino acid carbon skeletons can be used to make fatty acids. Fatty acids are made in the cytosol by a large complex of enzymesâfatty acid synthase:

Dimer of identical 260kDa subunits

Each monomer has three domains joined by flexible linker regions

Total of seven catalytic sites per subunit. The proximity of these sites allows intermediates to be handed efficiently from one active site to another without leaving the complex.

The reactions of fat synthesis are distinct from those of break down.

Fatty acid synthase is located in the cytoplasm (breakdown in mitochondrial matrix)

The intermediates of synthesis are covalently bound to the enzyme (rather than to CoA).

The committed step of fat synthesis is the carboxylation of acetyl-CoA to malonyl-CoA. This reaction is driven by ATP hydrolysis and thus, effectively, irreversible (Fig. 2.16).

Biotin is an essential co-factor for acetyl-CoA carboxylase

Allosterically activated by citrate (see below).

The reaction scheme is as follows (Fig. 2.17):

For the first round only, an acetyl-CoA is covalently linked to the acyl carrier protein (ACP), part of the fatty acid synthase protein monomer 1, via a flexible linker molecule (phosphopantetheine). It is then passed to the condensing enzyme (CE) in the other monomer (2)

Malonyl-CoA is covalently joined to the ACP of monomer 1

There follows a series of four reactions: condensation, reduction (with NADPH as the reductant), dehydration, and a final reduction (again using NADPH)

The elongated chain is transferred to the CE of monomer 1, and another malonyl-CoA is covalently linked to the ACP of monomer 2

Further rounds continue until a palmitoyl (C16) unit is formed

This is released by hydrolysis to give free palmitate.

Longer chain and unsaturated fatty acids are synthesized in the smooth ER.

>60% of fatty acids are >C18, with C20, C22, and C24 being the most common

Unsaturated fatty acids are also common

Catalysed by desaturase, cytochrome b5, and cytochrome b5 reductase

Most common in animals are the C16 palmotoleic and C18 oleic acids which have a single C=C bond at C9

As mammals cannot introduce double bonds past C9, such fatty acids have to come from the diet (essential fatty acids).

P.119The rate of synthesis and breakdown of fatty acids reflects the energy state of the cell.

When ATP levels in the cell are high, mitochondrial citrate rises as the ETC and the enzymes of TCA cycle are inhibited

Citrate leaves the mitochondria on a specific carrier in exchange for malate

In the cytosol, citrate is split into acetyl-CoA and oxaloacetate

Acetyl-CoA is converted into malonyl-CoA for fatty acid synthesis

Oxaloacetate is converted back into pyruvate

Pyruvate can return into the mitochondrion, where it is converted into oxaloacetate by pyruvate carboxylase

This process generates one NADH and one NADPH

Each cycle of the fatty acid synthase reaction results in the oxidation of two NADPH, the second of which comes from the pentose phosphate pathway (PPP; pp.138â9)

Acetyl-CoA carboxylase is regulated by phosphorylation

An AMP-sensitive kinase (AMPK) inactivates acetyl-CoA carboxylase when energy levels are low in the cell, thus inactivating fatty acid synthesis. This inhibition can be partially overcome allosterically by citrate. This effect of citrate is antagonized by high levels of palmitoyl-CoA, indicating an excess of fatty acids. Palmitoyl-CoA also inhibits the mitochondrial citrate exporter and the production of NADPH by the PPP

Fig. 2.19 Source of acetyl groups (acetyl-CoA) and reducing equivalents (NADPH) for fatty acid synthesis. The other NADPH comes from the PPP.

P.120 P.121 P.122 Ketogenesis by the liver With a balanced metabolism of carbohydrate and fat, the acetyl-CoA from Î²-oxidation will enter the TCA cycle to ultimately produce energy in the form of ATP.

During times of fasting and starvation, the liver maintains blood glucose levels by gluconeogenesis

Oxaloacetate from the TCA cycle is the starting substrate. The removal of this intermediate prevents acetyl-CoA from entering the TCA cycle (âfat burns in the flame of carbohydrateâ)

The build-up of acetyl-CoA leads to a greatly increased rate of formation of ketone bodies in the mitochondria (ketogenesis)

The major ketone bodies are acetoacetate and Î²-hydroxybutyrate (Fig. 2.20).

The liver cannot metabolize ketone bodies as it lacks the enzyme Î²-ketoacyl-CoA transferase, and so they enter the bloodstream

Ketone bodies are effectively a water-soluble, transportable form of acetyl groups

Heart, renal cortex, and adrenal glands all use ketone bodies as a preferred fuel source

The brain switches over to getting 50â75% of its energy needs from ketone bodies (rather then the usual glucose) after a few days of starvation

This reduces the gluconeogenesis load on the body, preserving protein (muscle) from breakdown

Ketone bodies are not only an efficient metabolic process (releasing almost as many ATP as acetyl-CoA entering the TCA cycle directly) but also provide a survival advantage to the tissues that receive them from the liver.

It is important to appreciate that animals cannot make glucose from acetyl-CoA.

Needs to combine with oxaloacetate to form any of the TCA cycle intermediates that can enter the gluconeogenic pathway. Therefore, no new intermediates are created (TCA cycle intermediates are essentially catalytic).

The levels of ketone bodies act as signals for availability of energy substrates.

High levels of acetoacetate acts as a signal for abundantly available acetyl groups. This inhibits the further breakdown of fat in adipose tissue.

Disease conditions can cause confused signals. Most common is diabetes mellitus (OHCM6 p.292).

Lack of insulin secretion means that the liver does not absorb glucose, and so the lack of carbohydrate leads to ketogenesis. This is made worse by the lack of signals to adipose tissue to inhibit fat breakdown

Ketone bodies (OHCM6 p.818) are acidic and their accumulation (up to 200-fold the normal concentration), and the ensuing metabolic acidosis, can be severe enough to impair CNS function

Acetoacetate is unstable and spontaneously decays to acetone. This can be smelt on the breath of uncontrolled diabetics.

Fig. 2.20 Ketone body production in the liver during excessive oxidation of fat in starvation or diabetes.

P.123 P.124 Integration As with all metabolic pathways, their regulation is the key to integrating their functions. Extrinsic regulation of fat metabolism is controlled by a number of hormones: insulin, glucagon, adrenaline, and thyroxine.

Lactate is the usual fate of pyruvate in red blood cells, which lack mitochondria and therefore have no ETC or TCA cycle.

P.131 P.132 Control of glycolysis Glycolysis is regulated by the energy needs of the cell. There are three main points of regulation. Hexokinase

High affinity (Km < 0.1mM) and shows strong end-product inhibition by glucose-6-phosphate (G-6-P) in most tissues

The inhibition by G-6-P is important as in the presence of high (glucose) and low rates of glycolysis, it prevents cellular depletion of Pi by hexokinase

Liver has glucokinase, which is lower affinity (Km ~ 7mM) and not inhibited by G-6-P

This is a problem in fructose intolerance as, in the absence of end-product inhibition, the liver generates large quantities of fructose-6-phosphate which cannot be metabolized further. This causes liver (ATP) to drop, compromising hepatocyte cellular function.

Phosphofructokinase (PFK)

PFK is the major site of regulation of glycolysis

There are several important allosteric regulators, both positive and negative.

Instead, increased amounts of pyruvate are converted to oxaloacetate, allowing the acetyl-CoA from fatty acids to be oxidized by the TCA cycle.

Pyruvate kinase

Strongly inhibited by ATP.

Different tissues express slightly different forms of the same enzyme (known as isozymes).

For example, hexokinase and glucokinase (above) are isozymes

This can be useful in diagnostic testing e.g. if a heart muscle enzyme isoform is found in the plasma it indicates that the individual has had a heart attack see (OHCM6 p.121) (p.18).

P.134 Use of other monosaccharides Although glucose is the major carbohydrate fuel, both galactose and fructose are important, with the latter making up a significant part of dietary carbohydrate. Galactose Galactose is metabolized by converting it into the glucose metabolite, glucose-6-phosphate (Fig. 2.22). This is a four reaction process.

Galactose is phosphorylated by galactokinase

Galactose-1-phosphate is converted into UDP-galactose by reaction with UDP-glucose, giving glucose-1-phosphate. This is then isomerized to glucose-6-phosphate

UDP-galactose is isomerized back to UDP glucose for re-use.

Galactosaemia is a rare, inherited inability to metabolize galactose.

A mild form is seen when galactokinase is deficient

In the severe form, galactose-1-phosphate uridyl transferase enzyme is absent

High blood and urine levels of galactose

Infants fail to thrive, with symptoms including:

Vomiting/diarrhoea after milk

Enlargement of the liver and jaundiceâeven cirrhosis

These are due to toxic effects of galactose-1-phosphate

Cataracts

Due to build-up of reduced form of galactose (galactitol) in the lens

Lethargy

Mental retardation (often delayed language skill acquisition)

Still persists even if patient has galactose-free diet.

Fructose Fructose has a more simple entry pathway into metabolism (Fig. 2.23).

In the liver, it is converted into fructose-1-phosphate by fructokinase

This is then split into dihydroxyacetone phosphate (DHAP) and glyceraldehyde

Glyceraldehyde is converted into glyceraldehyde-3-phosphate (GAP)

DHAP and GAP are both intermediates of the glycolysis pathway

In adipose tissue, hexokinase converts fructose into fructose-6-phosphate, which can continue through glycolysis.

Characterized by hypoglycaemia after fructose ingestion, and death in young children after prolonged ingestion

Fructose-1-phosphate accumulates intracellularly, effectively depleting the cells of free Pi and therefore reducing their ability to make ATP.

Fig. 2.22 Galactose metabolism.

Fig. 2.23 Fructose metabolism.

P.135 P.136 Aerobic oxidation of glucose Pyruvate dehydrogenase (PDH) is a key regulatory enzyme for aerobic oxidation of glucose, as it commits pyruvate to acetyl-CoA to enter the TCA cycle. Other potential fates include to lactate (anaerobic conditions), oxaloacetate (to replenish TCA cycle intermediates), or alanine (by transamination). The pyruvate dehydrogenase complex consists of a large number of subunits, with multiple copies of three catalytic and two regulatory enzymes, with five co-factors (all derived from water-soluble vitamins). Regulation is at two levels:

There is feedback inhibition by acetyl-CoA and NADH

However, more important is the regulation of the PDH enzyme complex by phosphorylation.

PDH is inactivated by phosphorylation

The kinase responsible for PDH phosphorylation is itself part of the PDH complex

The kinase is activated by acetyl-CoA and NADH

It is inhibited by CoASH, NAD+, pyruvate, and ADP

The phosphorylase that activates PDH is also part of the complex

It is Mg2+ and Ca2+ dependent. Ca2+ is important during muscle contraction, as it will cause the activation of PDH when energy is required

These regulatory factors allow PDH activity to reflect the metabolic state of the mitochondrion

An increase in the NADH/NAD+ or acetyl-CoA/CoASH ratio signals that the ETC is not operating fast enough to match NAD+ reduction to NADH. This could be due to lack of oxygen or a high ATP level (respiratory control is in operation) The result is an inactivation of PDH â a reduction in the rate of pyruvate entry into the TCA cycle.

The brain has a high energy requirement that is normally satisfied by aerobic glucose oxidation.

Anything that increases the oxidative stress in cells will then cause problems due to the lack of reduced glutathione. Examples include the antimalarial drug pamaquire and flavobeans (broad beans)

Symptoms include black urine, jaundice, haemolytic anaemia

NADPH is important in maintaining the erythrocyte membrane integrity

Deficiencies in G6PD lead to weakened cells that are more susceptible to haemolysis

There are over 300 known mutations in this enzyme

Frequency varies from <1% in Northern Europeans, 10% in Afro-Caribbeans, up to 25% in Southern Europeans

The high prevalence in Southern Europeans is due to its protective effects against malaria

Selective advantage may be due to malaria parasite needing PPP products and/or the extra stress caused by the parasite causing the red blood cell host to lyse before the parasite matures.

Fig. 2.24 Pentose phosphate pathway.

P.139 P.140 Storage of glucoseâglycogen breakdown and synthesis Glycogen is a readily mobilized storage form of glucose.

Glycogen is a very large, branched polymer of glucose

It has mainly Î±-1,4 glycosidic bonds, with branches about every tenth residue caused by Î±-1,6 bonds (p.34)

The many free 4-OH ends allow for rapid breakdown to release glucose.

There are separate pathways for breakdown (Figs. 2.25, 2.26) and synthesis of glycogen. Glycogen breakdown (glycogenolysis) Glycogen is broken down by the liberation of a glucose-1-phosphate (G-1-P) molecule, leaving the glycogen chain one residue shorter.

The reaction is catalysed by glycogen phosphorylase

The Î±-1,4 glycosidic bond is cleaved by phosphorolysis (cleavage of bond by orthophosphate), rather than by hydrolysis

In most tissues, G-1-P is converted into G-6-P by phosphoglucomutase, which can then enter the glycolytic pathway to form energy

As the main site of gluconeogenesis, the liver has the enzyme glucose-6-phosphatase. This converts G-6-P into glucose, which is released into the bloodstream.

Glycogen phosphorylase can only remove glucose residues from free chain ends until it is four residues from a branch point.

Three residues are moved by a transferase to an adjacent chain for future breakdown by glycogen phosphorylase

The remaining single residue is hydrolysed by Î±-1,6 glucosidase (debranching enzyme) to give glucose, leaving a linear chain for continued breakdown by glycogen phosphorylase

Debranching enzyme and transferase activity are present in the same 160-kDa polypeptide chain.

Fig. 2.25 Breakdown of glycogen is by the sequential liberation of a glucose-1-phosphate molecule.

Fig. 2.26 Schematic representation of glycogen breakdown, including of a branch point.

P.142 Regulation of glycogen synthesis and breakdown The two separate pathways of glycogen synthesis and breakdown must be regulated, both to maintain suitable plasma glucose concentrations and also to avoid futile substrate cycling. There are two potential forms of regulation: intrinsic and extrinsic.

Intrinsicâallows cells to respond to their own energy needs by breaking down glycogen when cell ATP and glucose levels fall, and to switch on glycogen synthesis when these concentrations rise

Extrinsicâmediated by hormones or other stimuli

Increases in intracellular levels of Ca2+ or cAMP will promote glycogen breakdown and inhibit synthesis e.g. to prepare muscle cells for action or liver to release glucose for other tissues

Insulin signals fed state and enhances glycogen synthesis and inhibits breakdown, thus storing energy for use in the future. These effects are mediated via reversible phosphorylation of the synthesis/ breakdown enzymes.

Regulation of glycogen breakdown The enzyme directly responsible for glycogen breakdown i.e. glycogen phosphorylase, can exist in two interconvertible forms:

The a form is active

The b form is usually inactive

The usually inactive b form can be converted into the active a form by phosphorylation

Catalysed by glycogen phosphorylase kinase

Glycogen phosphorylase a is deactivated by dephosphorylation by protein phosphatase 1 (PP1). This is the mechanism behind hormonally exerted extrinsic control

Although glycogen phosphorylase b is usually inactive, it can be activated allosterically by molecules that signal the energy charge of the cell

This represents the intrinsic control

In muscle cells, high (AMP) will activate glycogen phosphorylase b, whereas high (ATP) and (G-6-P) inactivate it. All three compounds act at the same allosteric regulatory site

The liver isoform of glycogen phosphorylase is different, in that active glycogen phosphorylase a is deactivated by the binding of glucose, but the b isoform is insensitive to AMP levels. This difference reflects the role of liver glycogen stores in supplying glucose for the rest of the body

Glycogen breakdown is prevented when plasma glucose concentrations are high.

What regulates the regulator (i.e. phosphorylase kinase)? Phosphorylase kinase is a very large protein (1200kDa), made up of (Î±Î²Î³Î´)4 subunits, and it can be controlled in two ways:

It is converted from a low to a high activity form by phosphorylation by protein kinase A (PKA)

As PKA is activated by cAMP, this is makes phosphorylase kinase sensitive to hormones such as adrenaline

Phosphorylase kinase is phosphorylated on a serine residue on subunits Î± and Î²

P.143

Phosphorylase kinase can be partially activated by Ca2+ at levels of ~1ÂµM because the Î³ subunit is calmodulin

This is important in muscle, where contraction is triggered by Ca2+ release from the SR

It will also make phosphorylase kinase sensitive to hormones which raise cytoplasmic Ca2+ (especially relevant in liver).

P.144 Regulation of glycogen production It is clearly important that glycogen synthetase is switched off when glycogen phosphorylase is activated and vice versa (i.e. that they are regulated reciprocally).

Glycogen synthase also exists in two formsâthe active a form and the inactive b form

Conversion from the active a to the inactive b form requires phosphorylation

The three most important kinases responsible are PKA, phosphorylase kinase, and the Ca2+-calmodulin CaM kinase II

Thus, the hormones which turned on glycogen breakdown will simultaneously turn off glycogen synthase:

Those which acted via CAMP, through activating PKA and phosphorylase kinase

There must be a cellular mechanism present to reverse the effects of the phosphorylation steps (i.e. activation of glycogen phosphorylase kinase and glycogen phosphorylase, and inactivation of glycogen synthase).

Protein phosphatase 1 (PP1) is the most important cell phosphatase regulating glycogen metabolism

PP1 has two subunitsâthe catalytic 37 kDa and the 160kDa glycogen-binding subunit

The glycogen-binding subunit is phosphorylated by PKA, rendering it unable to bind the catalytic subunit, thus inactivating it.

Further inhibition of PP1 is brought about by an inhibitor protein, known as inhibitor 1. When phosphorylated by PKA, this small protein blocks the catalytic subunit of PP1. Thus, cAMP not only activates the kinase cascade, but also prevents PP1 from dephosphorylating the enzymes involved in glycogen metabolism. What happens in times of plenty when glycogen synthesis needs to be switched on?

PP1 is activated by phosphorylation, and so glycogen synthase is dephosphorylated and activated. Simultaneously, glycogen phosphorylase kinase and glycogen phosphorylase will be dephosphorylated and inactivated

The net result will be increased glycogen synthesis and decreased glycogen breakdown.

Fig. 2.29 Regulation of the process of glycogen breakdown.

Fig. 2.30 Regulation of the process of glycogen synthesis.

P.146 Gluconeogenesis Gluconeogenesis (Fig. 2.31, 2.32) is the synthesis of glucose from non-carbohydrate precursors. This process plays different roles depending on the nutritional state and the tissue in question.

In tissues that are generating sufficient energy and have surplus nutrients, glucose is produced that can be stored as glycogen

The liver synthesizes glucose for export to other glucose-dependent tissues (especially brain, red blood cells) during starvation and intense exercise

Renal cortex also contributes about 10%

NB Mammals cannot convert fatty acids into glucose, as there is no enzyme to catalyse the reaction of acetyl-CoA into oxaloacetate

The glycerol backbone of triacylglycerols is a gluconeogenic substrate

The last unit of Î²-oxidation of a odd-chain fatty acid, propionyl-CoA, can also enter gluconeogenesis.

The gluconeogenesis pathway is not simply a reversal of glycolysis.

Thermodynamics favour glycolysis direction of glucose â pyruvate

There are three essentially irreversible reactions in glycolysis to be bypassed.

Fig. 2.31 Mechanism by which breakdown of muscle proteins supplies the liver with a source of pyruvate for gluconeogenesis during starvation.

Fig. 2.32 The complete gluconeogenesis pathway from pyruvate to glucose.

Trypsinogen is activated by enteropeptidase (enterokinase) released from the epithelial cells of the small intestine (enterocytes) and by active trypsin

The others are all activated by cleavage with trypsin

Exopeptidases remove the last (carboxypeptidase) or the first (aminopeptidase) amino acid from a peptide chain

Results in a mixture of amino acids and small peptides up to six amino acids long (oligopeptides)

Diseases which interfere with pancreatic secretion (e.g. pancreatitis, CFTR (OHCM6 p.178, p.478)) will prevent proper protein digestion and thus lead to protein malabsorption and malnutrition. This can be overcome by either supplying preparations of exogenous pancreatic enzymes or dietary supplements of easily digested proteins

The brush-border membrane of the enterocytes contain enzymes that continue digestion

Forms glutamate, which provides a pool of amino groups for making other non-essential amino acids or for deamination (see below)

Other amino group acceptors include pyruvate (â alanine) and oxaloacetate (â aspartate).

Glutamate is deaminated by glutamate dehydrogenase

The pooling of excess amino groups into glutamate means that only one deamination pathway is required

The deamination reaction regenerates Î±-ketoglutarate and a free ammonium (NH4+), plus an NADH

Glutamate dehydrogenase is allosterically regulated by increases in ADP and GDP. These compounds signal that amino acids need to be used as an energy source

The deamination reaction takes place in the mitochondria of liver cells

The major fate of NH4+ is incorporation into urea for excretion.

Other sites of ammonium production include:

Brain: breakdown (and therefore inactivation) of the neurotransmitter GABA to succinate and an ammonium ion

The ammonium ion is combined with Î±-ketoglutarate to produce glutamate, and then another ammonium ion is incorporated to form glutamine. This is transported to the liver for deamination and urea production

P.156 Tissue specific metabolism Not all tissues metabolize amino acids in the same way, and the metabolism by any one tissue often depends on the metabolic status of the body. Liver

The liver is the main site of amino acid degradation (deamination). It is also the major site of urea synthesis for nitrogen excretion

During fasting, the liver is the main site of gluconeogenesis, using carbon skeletons from amino acids

The liver plays a major role in the synthesis of the tripeptide glutathione.

Intestine

Enterocytes take up glutamine and release it as alanine. This enables them to generate energy from it (Fig. 2.34, p.153)

Enterocytes are the only cells to contain glutamate reductase, the synthetic enzyme for citrulline. Citrulline produced in the gut is metabolized to arginine in the liver. This arginine is converted to ornithine to increase the capacity of the urea cycle during periods of increased protein intake.

Skeletal muscle

During fasting and starvation, muscle protein is broken down so that the carbon skeletons can be used for gluconeogenesis by the liver

The main amino acids released are alanine and glutamine (Fig. 2.36)

Alanine is transported by the blood to the liver for deamination and gluconeogenesis

Glutamine is taken up by enterocytes for energy (above) and released as alanine.

Renal cortex

The renal cortex is the only tissue other than liver that can perform gluconeogenesis. It has a capacity of up to 10% of total glucose generation

The renal cortex converts citrulline via arginine to creatine, which is used by skeletal muscle to store high-energy phosphate bonds as creatine phosphate

Creatine phosphate spontaneously forms creatinine

Creatinine (OHCM6 p.684) is excreted by filtration by the kidneys, and its level in the blood can be used to assess renal function (OHCM6 p.272) (p.498)

The kidney is the major site of carnitine synthesis, with liver to a lesser extent

Carnitine is important in fatty acid metabolism (p.114).

Fig. 2.36 The glucoseâalanine cycle for transporting nitrogen to the liver as alanine, and glucose back to the muscles.

P.157 P.158 Cellular Organization of Metabolism Mitochondria Not only do the cells of different tissues have specialized metabolic roles, but also the different compartments in individual cells.

Mitochondria are the major site of energy (ATP) production in all cells except erythrocytes

TCA cycle, electron transport chain, oxidative phosphorylation all take place there

Mitochondria have their own separate genome (~4% of the total cell DNA)

The genome encodes 13 proteins, including some components of the electron transport chain and ATP synthase

Also 12S and 16S ribosomes and 22 unique tRNAs

Evolutionary origin of mitochondria suggests that they may have originally been free-living bacteria that became incorporated in cells in a symbiotic relationship (âsymbiontsâ)

Mitochondria can replicate in cells

They have their own protein-synthesizing apparatus

Mitochondrial density can vary both up and down e.g. up to 10-fold increase in resting skeletal muscle if it is repeatedly stimulated to contract over a prolonged period

Density also increases in hypoxia

Mitochondrial DNA is inherited almost exclusively from the maternal side as the egg has several hundred thousand molecules of DNA compared to only a few hundred in the sperm

Mitochondrial pathologies tend to be quite complex

Not all mitochondria are affected to the same extent, and there can be large variations in severity and time of onset of diseases

Eventually, the energy-generating capacity of the mitochondria falls below the level required to sustain the cellular function

The nervous tissue and heart are highly dependent on oxidative phosphorylation, and therefore most susceptible to mitochondrial mutations

The first disease discovered to be caused by mutations in mitochondrial DNA was Leber hereditary optic neuropathy (OHCM6 p.57), which causes blindness, with onset usually in adulthood (early to mid-life). Caused by mutations in the DNA for NADH-Q reductase (complex I)

In addition, the continued presence of developmental isoforms of cytochrome c oxidase (complex IV) in neonates can lead to severe respiratory distress or âfloppy baby syndromeâ. Recovery occurs after several months in a high-oxygen environment.

Wolmanâs disease: no detectable LAL activity â usually fatal by one year of age.

P.161 P.162 Peroxisomes

Peroxisomes (or microbodies) are small spherical or oval organelles with a fine network of tubules in their lumen

Over 50 peroxisomal enzymes have been identified

Some use or produce hydrogen peroxide (H2O2)âhence the name peroxisome

They play essential role in lipid breakdown (especially oxidation of very long chain fatty acids C24 and C26), bile acid synthesis, synthesis of glycerolipids, glycerol ether lipids (plasmalogens), and isoprenoids

Peroxisomes also contain enzymes for metabolizing D-amino acids, uric acid, and 2-hydroxy acids using molecular oxygen to form H2O2

Compounds are oxidized by the H2O2, which is itself then broken down to oxygen and water by catalase

By both forming and breaking H2O2 in the same organelle, the potential for cellular damage is limited

Peroxisome biogenesis disorders (PBDs) are rare and associated with insufficiencies in the peroxisomal enzymes

Tissues affected include liver, brain, kidney, skeletal system

Symptoms include low plasmalogens, high levels of very long chain fatty acids, and build-up of bile acid precursors

Most severe is Zellwegerâs syndrome (OHCM6 p.740)

Failure to traffic enzymes properly â non-functional peroxisomes; usually fatal by six months of age.

P.163 Protection of cells against reactive oxygen species

Peroxides are highly reactive oxygen species that can damage membranes and other biomolecules

As a protectant, cells contain a high level (~5mM) of the tripeptide, glutathione (GSH)

GSH is kept in its reduced form by glutathione reductase. The ratio of reduced glutathione (GSH) to oxidized (GS-SG) is ~500:1

The reduced SOD then reacts with a second superoxide and two protons to form hydrogen peroxide, reforming oxidized SOD: SODred + 2H+ + O2 â SODox + H2O2

The hydrogen peroxide formed by SOD is broken down by catalase: 2H2O2 â O2 + H2O

Antioxidant vitamins C and E are further cellular defences against oxidative damage

Being lipophilic, vitamin E is especially useful in protecting membrane lipids from peroxidation.

P.164 Integration and Regulation of Metabolism Overview Obviously not all of the metabolic processes that have been discussed in the preceding sections will be occurring at the same time in any one individual. Indeed, if this were the case, then there would be the danger of futile cycles that would be wasteful of energy. Therefore, it is important to have some concept of when and which pathways are largely active/inactive, and how this co-ordination of metabolism is brought about. The major situations that will be considered are:

Feeding

Starvation

The response to exercise

Pregnancy and lactation

Diabetes mellitus.

Cellular metabolic response to feeding Humans eat intermittently, so need to consume calories in excess of their immediate need and store energy for later in the form of glycogen and triacylglycerides.

In the affluent Western world, excess food consumption leading to obesity (OHCM6 pp.208â9) is the most common form of malnutrition.

What happens to the major digestion products of food (i.e. glucose, amino acids, and triacylglycerides) on ingestion? General points

Glucose and amino acids are taken to the liver by the portal vein before they enter the main circulation

Lipids are absorbed via the lymph system which drains into the vena cava i.e. they are not subjected to first pass metabolism by the liver

Lipids are transported in lymph/plasma as chylomicrons (p.547)

A rise in blood glucose triggers the release of insulin from pancreatic Î²-cells (p.584).

In the well-fed state Glucose

Taken up by the liver and stored as glycogen (glycogen synthesis, pp.140â1)

Glucose can be metabolized to pyruvate en route to fat synthesis

Triacylglycerides synthesized in the liver are carried in the blood in the form of VLDLs (p.28)

P.166 Cellular metabolic response to fasting and starvation In comparison to the fed state, when all tissues use exogenous glucose as a metabolic fuel, in a fasting state the body needs to use the energy it has stored as glycogen and fat. In starvation, protein will also be broken down to provide energy. General points

The pancreatic Î±-cells release glucagon, triggered by a fall in blood glucose

Skeletal muscle is unresponsive to glucagon

During fasting, the liver no longer uses glucose as a fuel source

During prolonged fasting/starvation, glucose use by other tissues falls as well

Within 24â36 hours, muscle has almost entirely switched to other fuel sources (fatty acids, ketone bodies), and the brain starts using ketone bodies

By ~3 weeks, the brain has largely switched to ketone bodies

Red blood cells, renal medulla, and, to a diminished extent, brain, are the only tissues still using glucose

Protein is not an inert energy store like fat or glycogenâbreakdown of proteins such as muscles and enzymes is a last resort of starvation.

Early fasting state

During the initial unfed state of fasting, liver glycogen is broken down (glycogenolysis) and glucose released into the circulation

This glucose is used by tissues such as the brain, red blood cells, and muscle

The alanine cycle becomes important

Alanine is generated in muscle cells by amination of pyruvate and released into the bloodstream

The alanine is taken up by the liver and deaminated; the nitrogen is excreted as urea, while the pyruvate is converted into glucose by gluconeogenesis

The Cori cycle operates

Similar to the alanine cycle, except involves lactate rather than alanine.

P.167 Later fasting state/starvation

In addition to being gluconeogenic, the liver becomes ketogenic and proteolytic (liver glycogen stores will have been exhausted)

Ketone bodies are formed from fatty acids released by lipolysis in adipose cells

Circulating fatty acids can be used directly as fuel by tissues e.g. muscle

Ketone bodies are used as fuel by brain and muscle

Glycerol from triacylglyceride breakdown used by liver for gluconeogenesis

Protein hydrolysis takes place in muscle, with alanine and glutamine being the main amino acids released

Alanine participates in the alanine cycle (see above)

Glutamine is metabolized by enterocytes, and alanine released

Glutamine is also an important fuel for cells of the immune system

Liver proteins are also hydrolysed, with the amino acids used as substrates for gluconeogenesis.

Energy is expended as âbasal metabolismâ (maintenance and repair of the organism), thermic effect of food (energy expenditure rises after meals), physical activity (âexerciseâ), and non-planned physical activity (âfidgetingâ). Typically, 5â10% of dietary energy is lost in faeces

Any difference between energy intake and expenditure is reflected in a change in the bodyâs energy store.

Positive energy balance (intake > expenditure) is a normal part of growth or anabolism e.g. during recovery from surgery or trauma. Positive energy balance beyond the needs of growth leads to fat accumulation and, ultimately, overweight and obesity (p.180).

Negative energy balance occurs during dieting or in anorexia, in people who become physically active for long periods (e.g. Mike Stroud and Ranulph Fiennes lost almost all their bodily energy reserves during their Antarctic crossing in 1992â3), and during periods of catabolism following major trauma or during severe infection.

Regulation of energy intake Energy intake is regulated by endocrine and neuroendocrine mechanisms.

Leptin is a peptide hormone secreted from adipocytes in response to the amount of fat stored

Leptin acts through hypothalamic receptors and a complex neuroendocrine system to reduce appetite. However, in humans this system is directed more towards avoiding starvation (leptin deficiency is associated with intense hunger)

Variation in leptin levels within the normal range seems not to have major effects on appetite

Treatment with recombinant human leptin has been remarkably successful in rare patients with complete leptin deficiency (characterized by massive childhood obesity, sexual immaturity, and T-cell dysfunction) but has little effect in the normal obese patient.

Energy intake is determined, to some extent, by diet composition. It is easier to overconsume energy when the diet is âenergy denseâ (high kJ per 100g).

Energy-dense foods are generally those with little water and a high fat or sugar content (âjunk foodsâ for instance)

Sedentary people have a PAL around 1.4; highly active people (e.g. soldiers in field training) have PALs up to 2.5; elite endurance athletes may maintain even higher PAL values e.g. 3â4 in Tour de France cyclists.

P.174 Nutrition We ingest nutrients that yield energy and nutrients that are essential for health.

Energy-providing nutrients are also called macronutrients (fat, carbohydrate, and protein)

Micronutrients include vitamins and minerals

The distinction between energy-yielding and other essential nutrients is not absolute. We need to ingest specific (âessentialâ) fatty acids and some essential amino acids. Water and oxygen are also essential but are not usually thought of as nutrients.

Nutritional disorders Nutritional disorders may involve a deficiency, an excess, or an imbalance of nutrients.

In developed countries, the most common nutritional disorder by far, in both humans and their pet animals, is obesity (OHCM6 p.466)

Under-nutrition is still prevalent in many parts of the world

This includes British hospitals: a number of surveys of in-patients have shown alarming degrees of malnutrition, especially in elderly patients. Poor nutrition in sick patients may increase morbidity and mortality.

Macronutrients (energy-yielding nutrients) Dietary fat

Fat is mainly ingested as triacylglycerol (triglyceride) (95% of dietary fat) but includes some phospholipids (4â5%) and cholesterol (typically 500mg/day)

Fat contributes an average of 35% of dietary energy in the UK

This has fallen over the last few years and is now in line with the recommendations of the UKâs Committee on Medical Aspects of Food Policy (COMA) made in 1994

The fatty acids of dietary fat may be saturated (typical of animal fat but present in all fats), monounsaturated (animal fat and vegetable oils, especially olive and rapeseed oils), or polyunsaturated (typical of sunflower and safflower oils; these are called n-6 or Ï-6 fatty acids)

The energy content is almost identical but the effects on serum cholesterol differ: saturated fat raises, monounsaturated and polyunsaturated fat lowers serum cholesterol

Average UK intake of saturated fatty acids (13% of dietary energy) is still greater than COMAâs recommendation of not more than 11% of energy

Complex carbohydrates are absorbed more slowly and, because they may be hydrated in foods (e.g. potato is 80% water), are low in energy density

Polysaccharides that are not digested in the small intestine (typically plant cell wall material) are classed as fibre or non-glycaemic carbohydrate

Bacterial fermentation in the colon produces gas and short-chain fatty acids that may have beneficial effects on colonic function including cancer protection.

Dietary protein Most people in the Western world are not short of protein.

Typical protein intake is 60â100g/day, whereas 40â50g/day is probably sufficient for life

Vegetarians not taking a range of protein sources may be at risk of deficiency of particular essential amino acids. Wheat (low in lysine) and legume protein (low in cysteine, methionine) complement each other.

Micronutrients Micronutrients have diverse functions in the body. Anyone eating a balanced and varied diet that meets energy requirements is unlikely to suffer from vitamin deficiencies and, for most people, there is no clear evidence of benefit from supplementation. There are exceptions.

High folic acid (OHCM6 p.632) intake in the periconceptual period reduces neural tube defects in babies and fortification of flour with folic acid is mandatory in the US and Australia (not yet in Europe)

Women with heavy menstrual losses may be iron deficient

However, in the developing world it is estimated that billions of people suffer from micronutrient deficiency (iron, zinc, vitamin A)

Some vitamins (A, D, E, and K) are fat-soluble and are only absorbed in the presence of dietary fat

Intrinsic factor is secreted from the gastric mucosa and impaired secretion will result in vitamin B12 deficiency and pernicious anaemia (OHCM6 p.634)

In some parts of the world there are mineral deficiencies in the soil e.g. selenium in China, some parts of the US, and Finland; iodine in many upland areas of Africa, Asia, and South America. Public health campaigns are then needed to encourage supplementation.

P.176 Assays: diagnostic enzymology Measurement of enzyme activity plays an important and continually increasing role in medical diagnosis. There are basically two applications of diagnostic enzymologyâmeasurement of the enzyme itself, or exploitation of enzyme specificity to measure the concentration of its substrate. Enzyme activity measurement This is important in two areas: assessment of tissue damage (in which enzymes from the affected tissue are released into the circulation) and diagnosis of specific enzyme deficiencies.

In the case of tissue damage, the enzyme is being measured in an abnormal site, usually in serum, and results are dependent on the half-life of the protein after release from the cells of origin

Enzymes released from tissues are cleared relatively rapidly from the circulation (half-lives of the order 10â20hours) and this provides current information about the damage

Many enzymes are present in a variety of different tissues and simple measurement of a single enzyme may not be sufficient to identify the site of origin

Measurement of several different enzymes or additional analysis of isoenzyme forms which may be preferentially expressed in particular tissues may provide the necessary specificity

Isoenzyme forms of lactate dehydrogenase, creatine kinase, and alkaline phosphatase are most widely used in this regard (OHCM6 p.704).

Enzyme activity is determined with saturating substrate concentrations and can be measured by following the rate of change in the concentration of substrate or product or by measuring the amount of substrate remaining or product formed after a specified time

To avoid problems due to substrate depletion, reverse reaction, and product inhibition, conditions should be chosen to measure the initial rate, and all relevant conditions such as temperature and buffer composition and pH should be standardized

In some cases, it is necessary to couple the enzyme reaction being measured to a second enzyme-catalysed reaction in order to generate a readily detectable product

In this situation, it is necessary to ensure that the coupled enzyme and any additional substrates are not rate-limiting.

For the investigation of specific enzyme deficiencies, the same technical considerations are relevant

However, these investigations are required much less frequently and are usually performed in specialist centres with the necessary experience and expertise

While enzyme measurement for assessment of tissue damage is usually performed on serum samples, diagnosis of enzyme defects requires direct analysis of affected tissues

Some defects are expressed in readily accessible samples, such as blood cells or cultured fibroblasts, but others may require tissue biopsy

P.177

With increasing identification of the genes responsible for many enzyme deficiencies, direct mutation analysis, avoiding the need for biopsy and complex enzymological studies, is becoming more widely available.

Measurement of metabolite concentrations using enzymes The specificity of many enzyme-catalysed reactions can be exploited to measure the concentration of their substrate. This is particularly important when dealing with the complex mixtures of related compounds found in biological samples such as blood or urine.

For example, there are several enzymes which oxidize glucose with the necessary specificity for measuring the blood glucose concentration

For this type of analysis, it is important that the equilibrium of the reaction lies far to the right and that there is sufficient enzyme to ensure almost complete conversion of substrate to product within a short time.

P.178 Inborn errors of metabolism There are over 300 defined enzyme deficiencies affecting the function and regulation of many different metabolic pathways.

Most present in the first years of life; many within the newborn period

Although identification of specific enzyme deficiencies is performed by a small number of specialist laboratories, clues to the nature of the underlying biochemical defect can usually be found on the basis of a number of widely available screening tests.

Inborn errors of metabolism can be divided into three main groups based on the consequences of the biochemical defect, which in turn is related to the type of metabolic pathway involved. Group 1 Conditions due to accumulation of toxic metabolites. These include the disorders of amino acid oxidation and the related organic acidurias, defects of the urea cycle, and the various forms of carbohydrate intolerance. Clinical presentation

The reaction to toxic intermediates is non-specific

Most patients present in the newborn period, but there may be delayed onset, often with milder symptoms

Clues to the diagnosis are a period of normality after birth before symptoms commence, precipitation of symptoms by feeding or intercurrent illness, and a positive family history

A small number of inborn errors fulfil criteria for universal newborn screening. However, this is widely performed only for phenylketonuria

When there is already an affected child in a family, early diagnosis in subsequent siblings, before the onset of symptoms, is associated with much better prognosis. Specific tests will be indicated depending on the prior diagnosis

In all cases, rapid assessment is essential once symptoms of toxicity appear

The most widely available and useful screening tests are for urine organic acids and urine and plasma amino acids

Neurological dysfunction is again prominent, but there may also be specific problems related to energetic failure in other organs such as heart, liver, kidney, and skeletal muscle.

P.179 Investigation

Most patients will be screened as for patients in group 1, although blood lactate and pyruvate concentrations are often more relevant, and the lactate concentration in cerebrospinal fluid may be particularly helpful in patients with predominantly neurological dysfunction

Fasting tests and muscle biopsy for morphology and enzymology may be indicated, but are usually performed in specialist units.

Defects in metabolism of macromolecules These include the lysosomal storage diseases and peroxisomal diseases. Clinical presentation

These conditions are generally of later onset and are often characterized by organomegaly, with or without progressive neurodegeneration

Appearance of features such as characteristic facial appearance, skeletal deformities, and corneal clouding may provide additional clues.

Investigation

Apart from analysis of urine glycosaminoglycans for the diagnosis of the mucopolysaccharidoses, there are no screening tests for this group of conditions

Vacuolated lymphocytes or foamy macrophages in bone marrow may provide a clue to a lysosomal storage disease, and plasma very long chain fatty acids are often elevated in peroxisomal diseases. However, diagnosis is usually based on specific enzyme assays

Once an inborn error of metabolism has been suggested on the basis of the tests outlined above, further investigation and management is generally undertaken by specialist centres where expertise in laboratory diagnosis, monitoring, and long-term treatment (where possible) is available

As many inborn errors of metabolism present with non-specific symptoms which can mimic other conditions, such as infection, the key role for the primary physician is to suspect that an inborn error may be the cause of the patientâs problems and to arrange for the appropriate screening and routine laboratory tests to be performed as quickly as possible

While the course of many inborn errors of metabolism is unaltered by any therapy, there are continuing advances in management, especially of conditions due to accumulation of toxic metabolites

It is in this group, particularly, that delayed diagnosis may result in a much poorer prognosis, with a high risk of permanent brain damage.

P.180 Obesity and treatment Obesity is an excessive accumulation of body fat.

A useful measurement is the body mass index (BMI) (OHCM6 p.208):

A BMI of 25â30kg/m2 is usually taken to represent overweight; a BMI >30kg/m2 obesity

However, a weight lifter (for instance) may have a BMI of 30kg/m2 without excessive fat accumulation

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